Transformation of agricultural, industrial and city wastes is one of the most delicate problems to be solved at present. Hitherto made attempts have been aiming at the solution of particular cases. Moreover, the biotechnology applied to this field has not given any expected result so far because, even if genetically modified microorganisms having the capability of degrading the poison and yet harmful waste materials have been developed, the use of such microorganisms in agriculture or in decontamination operations is subjected to the release thereof into the environment, which is causing many problems also due to the risk of transferring the modified genetic characteristics to other organisms.
In contrast, there is provided according to the present invention the use of one or more enzymes bound on materials which are not expensive, becomes integrated in the application environment, and do not affect but positively modify the catalytic activities by protecting them with the time and in the operation fields. The use of such bound biological catalytic activities, even if they are obtained from genetically modified microorganisms, does not imply the above mentioned problems nor meet with application difficulties.
Several techniques of binding enzymes and cells have been known for many years, such techniques being aimed at the binding or localization of such enzymes during continuous catalytic processes.
The purpose of such binding techniques has been achieved either by covalent bonding with insoluble or made insoluble "functionalized" polymers, or by the absorption on organic or inorganic insoluble substrates, or by the entrapping within gelatinous matrices or semipermeable microcapsules.
The essential reasons leading to the production and the use of bound enzymes are three: the first one is that the enzymes can have in this way a considerable operative advantage over the free enzymes; the second one is that the chemical and physical characteristics of the enzymes can be selectively modified; the third one is that the enzymes can act as models of natural systems and enzymatic activities bound to the walls or to cellular organelles.
Such preparations have the operative advantages of being re-usable as well as being used both in static and dynamic systems, interrupting immediately and totally any catalytic reaction, forming under control the requested products, allowing simpler industrial plants of different types to be designed as high operating temperatures and pressures are not requested, and finally achieving a greater efficiency in those processes requiring several reactions.
Changing or at any rate modifying the reactivity of the enzymes is bound to the binding process, especially if the latter is a chemical process. In fact the methods based upon a chemical binding process depend on the reactivity of some functional groups of the enzymes. As such functional groups are eliminated or changed into groups having different structures and reactivity, the chemical-physical characteristics of the enzymes bound by such technique can be subjected to change deeply.
Cross-linking methods are based upon the covalent bonding of the molecules of the enzyme and bi- or multi-functional reactants so that threedimensional reticular aggregates completely insoluble in water are formed without using insoluble substrates. The method provides the addition of an appropriate quantity of cross-linking agent to an enzymatic solution under the most suitable conditions to form the insoluble derivative. The best conditions to achieve a good insolubility and to maintain the enzyme activity unchanged are unfortunately determined each time by a series of experimental tests. Such reactants are, for example: compositions including carbonyl groups adapted to react with the amino-group of L-lysine, L-histidine, L-tyrosine, L-arginine, L-cysteine; isocyanate adapted to react with the primary aminogroups; iodoalkanes adapted to react with the nucleophilic groups; iodoacetamides adapted to react with L-cysteine. Among such compositions the glutaraldehyde is mostly used. The best conditions to achieve a good insolubility efficiency and to maintain at the same time a considerable enzyme activity depend on the balance of factors such as concentration of the enzyme and the cross-linking agent, pH, ionic force, temperature, and reaction time. The disadvantages of such preparation method essentially consist both in the difficulty of controlling the intermolecular cross-linking which can form large enzyme aggregates having a high activity and in that a gelatinous structure is formed which cannot assure a good mechanical resistance and then a good flow characteristic in the continuous processes.
The physical absorption of the enzyme is carried out by mixing it with the substrate under suitable conditions. Subsequently, after a convenient period of time is elapsed, the enzymatic insoluble compound is separated from the starting material by centrifugation or filtration. As no chemical reaction occurs in such method of preparation, the composition of the enzymes does not change. The absorption depends on variables such as pH, solvent, ionic force, enzyme concentration, absorbent quantity, temperature. The greatest influence on the enzyme absorbed on a solid substrate is due to the concentration of the enzyme exposed per surface unit of the substrate during the linking process. The main drawback of such preparation method is that the bonding forces between enzyme and substrate are generally weak so that the enzyme can be desorbed in use. As a consequence, the mean life of such preparations may be short so that they cannot be used in long-time processes. The generally used substrates are: alumina, activated coal, clays among which bentonite, collagen, glass, hydroxylapatite and diatom dust are mainly used. Enzymes can also be bound by physical absorption onto materials having some affinity therewith, which is achieved by means of a derivatization of the substrate.
Methods leading to the ionic bonding are mainly based upon ionic interactions between the enzymatic protein and the solid substrate having residues adapted to exchange ions. Bonds due to the Van der Waals forces and ionic bonds cooperate to the formation of the resulting compounds. The main difference between such preparation methods and those based upon the physical absorption of the proteins consists in the bonding force between enzyme and substrate. As in the case of the preparation of the absorption compounds, also in the above preparation the compound is formed by contacting the substrate with the enzyme solution under softer reaction conditions than those of covalent bonding. The used substrates are generally polymers or compositions used in ion-exchange chromatography; they are organic substrates including residues adapted to exchange cations and anions. Also inorganic substrates, especially silica, to which ionic residues have been applied, can be used. Generally, the organic substrates are derivative of polysaccharides, in particular cellulose and dextrans, and polystyrene polymers. The compositions used to form the ion-exchanger substrates include: anion-exchanger compounds such as amino compounds, guanidine compounds, a.s.o. (for example, DEAE-, TEAE-, ECTEOLA-compounds), and cation-exchanger compounds having sulfuric, phosphoric and carboxylic groups.
The complexing with transition metals is based upon the chelating properties of the transition metals, in particular titanium and zirconium. The enzymes are made insoluble by activation of molecular groups at the surface of some substrates through such metals. The nucleophilic groups (--OH, --NH.sub.2, --SH, a.s.o.) are good bonding agents for the transition metals, and therefore such metals can carry out the complexing both of the polymer and the enzyme. Substrates such as cellulose and silica have hydroxil groups acting as bonding agents adapted to substitute other agents. For such reason enzymes having in their molecule the alcoholic hydroxyl groups of L-scrine and L-threanine, the free sulfhydryl groups of L-cysteine, and the .epsilon.-aminogroups of L-lysine can be bound by such method. Besides organic substrates such as cellulose and chitin also inorganic substrates such as silica and glass wool have been used but in the latter case the results have not ever been good. Generally, such method allows high specific activities of the compounds to be held, however, the continuous activity of such compounds has a variable stability.
The covalent bonding for binding enzymes to organic and/or inorganic substrates is the most complex among the techniques leading to the bonding between the two above mentioned components as the reaction conditions for the bonding are generally harder and the manipulations to be carried out are more complicated. What is important in such preparations is that the aminoacids of the enzymes essential for the catalytic activity are not involved in the bonding with the substrate. Sometimes the protection has been obtained by the addition of the substrate or a competitive inhibitor of the enzyme activity to the reaction mixture. The main factors which have to be considered in the preparation of the bound enzymes by means of a covalent bond are three: the functional groups of the proteins which can form the covalent bond under soft conditions; the kind of reaction between enzyme and substrate; the substrate to which a functional group adapted to bind the enzyme has been added. The easiest way for obtaining insoluble derivatives of enzymes and substrates is that the enzyme react in a solution upon the substrate already including the suitable reactants but this is only seldom possible because the substrate does not generally include them. In fact the substrate materials which are mostly used include in most cases hydroxyl groups, carboxyl groups, amino groups, and amide groups, and these groups should be activated by preliminary reactions with the aminoacid residues of the enzymes.
The entrapping methods are based upon the mixing of the enzymes with the latex of a polymer matrix or upon the storing thereof within semipermeable membranes having pores which are small enough to prevent proteins from going lost and at the same time large enough to allow substrates and products to pass through. Such methods can be applied to any enzyme having not very neutralized activities at least in comparison with the methods requiring chemical reactions.
Gel entrapping methods are based upon the enzyme entrapping within the interstitial spaces of cross-linked polymer gels insoluble in water. The polymer reticulum can be obtained by monomer, oligomer or polymer precursors by changing the solubility variables such as solvent, temperature, ionic force, and pH.
A different binding method leading to the entrapping of the enzyme into micro-cavities is the physical entrapping of an enzyme by dissolving a polymer adapted to form fibers (for example cellulose triacetate) in an organic solvent insoluble in water (for example, chloroform, methylene chloride, carbon tetrachloride) and by emulsifying such solution with the acqueous solution of the enzyme. The solution is poured in a liquid coalescent (for example, toluol or petroleum ether) which precipitates the emulsion under the form of a filament including microdrops of enzymatic solution. However, such preparation is limited to enzymes acting on substrates having a low molecular weight because of the difficulty due to the steric hindrance of the large substrates. Other problems originate from the necessity of using liquids insoluble in water as solvent of the polymer.
The enzymes can also be entrapped within microcapsules prepared along with organic polymers. The membranes enclosing the enzymes are semipermeable in comparison with substrate and products. The advantages of such method essentially consist in that the surface area exposed to the reaction is high and the volume in which the reaction occurs is small, that several enzymatic activities can be bound at the same time, and that the enzymes can be kept in their soluble state. The main disadvantages consist, however, in that such method cannot be applied to enzyme activities which catalyze reactions on substrates of high molecular weight, and that the disactivation may occur during the preparation.